DNA Quantification

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== Overview ==
== Overview ==
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This protocol uses a spectrophotometer to quantify the amount (μg/mL) of DNA and then uses a simple equation to convert this mass concentration into a molar concentration.  The molar concentration is much more useful for most enzymatic processes.   
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This protocol uses a spectrophotometer to quantify the amount (μg/mL or ng/μL) of DNA and then uses a simple equation to convert this mass concentration into a molar concentration.  The molar concentration is much more useful for most enzymatic processes.   
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*'''Example:''' digesting 500ng of a 2KB plasmid is twice as much work as digesting 500ng of a 4KB plasmid with the same multiple cloning site.
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*'''Example:''' digesting 500ng of a 2KB plasmid is twice as much "enzymatic work" as digesting 500ng of a 4KB plasmid with the same multiple cloning site.
== Procedure ==
== Procedure ==
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#Dilute the DNA sample 30X by combining the following in a cuvette
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1. Get DNA by any means necessary.
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:*87µl water
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<br>
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:*3µl DNA prep
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2. Run the DNA quantification (260/280) test on a spectrophotometer.
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#Run the DNA quantification (260/280) test on the spectrophotometer with a dilution of 30.
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*Be sure blank a sample first.
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#Make sure that the A260 measurement is between 0.1 and 1.
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*You can use only 1μL of sample if you use a NanoDrop
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#If is too low then repeat the measurement using 15X dilution.
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*Or you can use 90μL of diluted sample using a UV cuvette
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:*84µl water
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::1. Dilute the DNA sample 30X by combining the following in a cuvette:
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:*6µl DNA prep
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:::*87µl water
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#Calculate the molar concentration of DNA using the following equation.</br>
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:::*3µl DNA prep
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::2. Run on the spectrophotometer with a dilution of 30.<br>
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::3. Make sure that the A260 measurement is between 0.1 and 1.<br>
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:::*If is too low then repeat the measurement using 15X dilution.<br>
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::::*84µl water
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::::*6µl DNA prep
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3. Calculate the molar concentration of DNA using the following equation:
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<br>
*Picomoles/µl =  DNA Concentration(µg/ml) / [0.66*DNA Size(bp)]
*Picomoles/µl =  DNA Concentration(µg/ml) / [0.66*DNA Size(bp)]
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4. Or calculate the μL of dna to add to obtain a desired molar amount of DNA.<br>
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*μL = Picomoles*[0.66*DNA Size(bp)]/DNA Concentration(µg/ml)
== Notes ==
== Notes ==
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*One mole of single base pairs weighs 660 grams.
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**One picomole of 1000bp weighs 660ng.
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* 1ug = 1000ng
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* 0.001ug = 1ng
==References ==
==References ==
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"Convert ng to ug - Conversion of Measurement Units." http://www.convertunits.com/from/ng/to/ug
[[Category:Protocol]] [[Category:DNA]] [[Category:In vitro]]
[[Category:Protocol]] [[Category:DNA]] [[Category:In vitro]]

Current revision

back to protocols

Contents

Overview

This protocol uses a spectrophotometer to quantify the amount (μg/mL or ng/μL) of DNA and then uses a simple equation to convert this mass concentration into a molar concentration. The molar concentration is much more useful for most enzymatic processes.

  • Example: digesting 500ng of a 2KB plasmid is twice as much "enzymatic work" as digesting 500ng of a 4KB plasmid with the same multiple cloning site.

Procedure

1. Get DNA by any means necessary.
2. Run the DNA quantification (260/280) test on a spectrophotometer.

  • Be sure blank a sample first.
  • You can use only 1μL of sample if you use a NanoDrop
  • Or you can use 90μL of diluted sample using a UV cuvette
1. Dilute the DNA sample 30X by combining the following in a cuvette:
  • 87µl water
  • 3µl DNA prep
2. Run on the spectrophotometer with a dilution of 30.
3. Make sure that the A260 measurement is between 0.1 and 1.
  • If is too low then repeat the measurement using 15X dilution.
  • 84µl water
  • 6µl DNA prep

3. Calculate the molar concentration of DNA using the following equation:

  • Picomoles/µl = DNA Concentration(µg/ml) / [0.66*DNA Size(bp)]

4. Or calculate the μL of dna to add to obtain a desired molar amount of DNA.

  • μL = Picomoles*[0.66*DNA Size(bp)]/DNA Concentration(µg/ml)

Notes

  • One mole of single base pairs weighs 660 grams.
    • One picomole of 1000bp weighs 660ng.
  • 1ug = 1000ng
  • 0.001ug = 1ng

References

"Convert ng to ug - Conversion of Measurement Units." http://www.convertunits.com/from/ng/to/ug

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